EP1429808A1 - Solid composition containing bacillus-type non-pathogenic bacterial spores - Google Patents

Solid composition containing bacillus-type non-pathogenic bacterial spores

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Publication number
EP1429808A1
EP1429808A1 EP02791476A EP02791476A EP1429808A1 EP 1429808 A1 EP1429808 A1 EP 1429808A1 EP 02791476 A EP02791476 A EP 02791476A EP 02791476 A EP02791476 A EP 02791476A EP 1429808 A1 EP1429808 A1 EP 1429808A1
Authority
EP
European Patent Office
Prior art keywords
spores
composition
composition according
billion
bacillus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP02791476A
Other languages
German (de)
French (fr)
Other versions
EP1429808B1 (en
Inventor
Tiziano Prato
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi Aventis SpA
Original Assignee
Sanofi Synthelabo OTC SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanofi Synthelabo OTC SpA filed Critical Sanofi Synthelabo OTC SpA
Priority to SI200230908T priority Critical patent/SI1429808T1/en
Publication of EP1429808A1 publication Critical patent/EP1429808A1/en
Application granted granted Critical
Publication of EP1429808B1 publication Critical patent/EP1429808B1/en
Priority to CY20101100602T priority patent/CY1112608T1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1611—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds

Definitions

  • the present invention relates to a solid composition containing spores of non-pathogenic bacteria, for use in the pharmaceutical, veterinary or nutritional fields, in particular a composition containing spores of bacteria of the genus Bacillus.
  • compositions based on spores are known. They are produced according to different techniques, such as, for example, lyophilization or "spray drying", as are known liquid suspensions of spores or simple mixtures of spores with conventional excipients in the pharmaceutical technique, such as by example WO 99/49877, which describes compositions of bacillus spores producing lactic acid for the reduction of cholesterol.
  • compositions of the prior art Another drawback of the compositions of the prior art is the low concentration of active principle, therefore of spores, which can be introduced into the composition itself.
  • Liquid compositions based on spores are available on the market, for example the suspension of bacillus spores marketed in Italy for a long time under the brand name “EnterogerrninaV. As long as it is effective and appreciated for a long time by consumers, this suspension cannot not contain a very high concentration of active ingredient (greater than 2 billion spores / 5 ml).
  • the object of the present invention is to provide a solid composition based on spores of non-pathogenic bacteria, hereinafter simply called "Spores", which is simple to produce, easy to store and which contains a high quantity of active principle and stable over time.
  • Composition makes it possible to fix a large quantity of spores to the matrix by creating a solid composition at high concentration whose particle size and specific surface make it particularly suitable for the in vivo release of spores throughout the gastrointestinal tract.
  • the composition has excellent fluidity properties and, therefore, it is capable of being industrially treated for the purposes of its formulation in single-dose or multi-dose compositions, without it being necessary to subject it to further processing.
  • the invention relates to a composition of spores of non-pathogenic bacteria of the genus Bacillus adsorbed on a matrix formed of at least one compound insoluble in water and of a cellulose derivative, capable of being obtained. by the fluidized air bed technique.
  • the invention relates to a composition of spores of non-pathogenic bacteria of the genus Bacillus capable of being obtained by a process which comprises treating according to the technique of the fluidized air bed a liquid suspension of said spores with a matrix formed of a water-insoluble adsorbent compound and a cellulose derivative.
  • the expression “adsorbent compound” designates any chemical compound, or a mixture of chemical compounds, which can be ingested and which has adsorbent properties; preferably, the water-insoluble adsorbent compound is chosen from the group formed of clays, kaolin, calcium carbonate, colloidal silicas, magnesium silicate and aluminum and derivatives of cellulose and bentonite, kaolin being particularly advantageous .
  • cellulose derivative designates any cellulose derivative which can be ingested, such as, for example, microcrystalline cellulose, methylcellulose, hydroxypropyl- methyl cellulose, etc. of which wide commercial ranges are available, microcrystalline cellulose being particularly preferred.
  • spores By the expressions “adsorbed spores”, “adsorption” or “adsorb” means, according to the present invention, the retention of spores on the surface of the matrix from which said spores are releasable in the digestive tract.
  • the spores can be retained on the matrix by any attachment, namely by any possible link (chemical, biological, physical, etc.) depending on the type of matrix used.
  • the relative amounts of the two components that make up the matrix can vary within a wide range.
  • the weight ratio between the adsorbent compound and the cellulose derivative can be between
  • the matrix of the invention is a mixture insoluble in water, inert towards the spores, where by "inert towards the spores" is meant that it does not interfere negatively with the spores.
  • the spores which can be used in the present invention are preferably spores of non-pathogenic bacteria which are particularly useful in the pharmaceutical, veterinary and / or nutritional fields.
  • the composition of the present invention may contain spores of a single Bacillus or spores of different Bacilli mixed.
  • the composition of the invention comprises spores of Bacillus subtilis or Bacillus clausii.
  • the composition of the invention comprises the spores of one or more strains of Bacillus clausii (whose previous taxonomic name was Bacillus subtilis), deposited in accordance with the Treaty of
  • an aqueous suspension containing the spores hereinafter called “concentrated suspension” is sprayed onto a matrix obtained by mixing at least one adsorbent compound and a cellulose derivative, constantly agitated by a flow of air throughout the duration of the process, in a tool provided for this purpose, such as, for example, a machine for granulating with a fluidized air bed.
  • the concentrated suspension is aqueous which has a very high concentration of spores.
  • the concentrated suspension is obtained by inoculation of one or more strains of Bacilli in a culture medium (for example based on peptones and mineral salts), by incubating the mixture at an appropriate temperature for 48-72 hours under aerobic conditions, by centrifuging the cells from the spent medium with distilled water and then pasteurizing the suspension obtained.
  • a culture medium for example based on peptones and mineral salts
  • the concentrated suspension thus prepared has a Bacillus spore concentration greater than 10 billion per gram, normally between about 15 and about 25 billion per gram or even more.
  • the concentrated suspension is preferably stored after being frozen because of its high instability at room temperature.
  • the concentrated suspension will be thawed just before its use in the process of the present invention.
  • the concentrated suspension can be an extemporaneous suspension of spores preserved in lyophilized form in water.
  • the temperature of the air flow used in the process of the present invention is between ambient temperature and the maximum temperature supported by the spores; according to a preferred aspect, the process takes place preferably at a temperature between 40 ° and 90 ° C, advantageously between 60 and 80 ° C.
  • the composition obtained is subsequently kept in suspension by means of the flow of heated air until it has reached the desired residual humidity, preferably up to the humidity is less than 3%, advantageously less than or equal to 2%.
  • the duration of the process is defined by the quantity of concentrated suspension to be sprayed, by the spraying speed as well as by the temperature of the air flow. Normally, for the treatment of quantities around 30 kg of matrix, approximately two hours are necessary to complete the process.
  • the process for preparing the composition constitutes a further subject of the present invention.
  • the composition of the invention can be obtained in a highly concentrated form and have a concentration of, for example, between 3 and 30 billion spores per gram of final composition, for example between 5 and 20 billion spores per gram of final composition, advantageously about 10 billion spores per gram, thus allowing the administration of consistent amounts of spores in small volumes.
  • This important property makes the composition particularly easy to use and, for example, can be introduced into small capsules or sachets or incorporated into food or other compositions. If necessary, the composition can also be administered after being resuspended in water or other suitable liquids.
  • composition of the invention has been found to be stable while retaining its title unchanged for a long time, even at temperatures above ambient temperature (approximately 40 ° C.).
  • the determination of the spore titer of the composition of the invention can be carried out according to any procedure, for example by counting on conventional culture plates.
  • composition of the invention has a large specific surface, thanks to a very fine particle size, until it reaches
  • composition has no odor or flavor and can thus be added to foods, drinks, or other compositions without altering their original flavor.
  • the composition may contain additives suitably chosen according to the final consumer, the method of absorption or the type of subsequent treatment to which it is desired to subject it, on the sole condition that the additives are inert versus the spores.
  • additives suitably chosen according to the final consumer, the method of absorption or the type of subsequent treatment to which it is desired to subject it, on the sole condition that the additives are inert versus the spores.
  • magnesium stearate and / or microcrystalline cellulose For example, if it is desired to introduce the composition into hard gelatin capsules, it could be useful to add magnesium stearate and / or microcrystalline cellulose.
  • flavoring agents may be added which may impart particular fragrances or flavors to the composition. Any subsequent components can be added to the matrix before adsorption of the spores or simply to the final composition obtained by the process of the invention.
  • the composition can be subject to subsequent modifications; the composition can therefore be granulated according to well known techniques if it is desired to compress it, or be treated so as to obtain controlled-release compositions according to well known techniques, in order to modify the time of its release in the intestine.
  • composition can be administered in varying amounts depending on the needs for which it is administered. Generally, when administered to humans, one can provide 10 billion spores / day and even more, advantageously from 1 to 8 billion per day, for example 2, 4 or 6 billion spores per day , administration being possible in a single take or repeatedly.
  • composition of the invention can, where appropriate, be formulated in dosage units; for example, thanks to its qualities, it can be easily formulated in gelatin capsules, such as capsules in the 0, 1 or 2 format, chosen by the sector expert according to the dosage.
  • the dosage units in the form of capsules or sachets, containing the composition of the invention represent a further object of the present invention.
  • these dosage units can contain from 1 to 10 billion of said spores, advantageously from 2 to 5 billion, from 50 to 500 mg, for example from 50 to 250 mg of kaolin and for example from 50 to 600 mg, for example from 50 to 300 mg of microcrystalline cellulose.
  • These dosage units can be administered one or more times per day, depending on the need and concentration of the dosage unit.
  • compositions of the invention packaged in glass or in polyethylene were subjected to stability studies to evaluate their behavior at different temperatures (from 5 ° C. to 40 ° C.) and degrees of humidity ( up to 75% relative humidity). The results after 24 months showed that the titer of the composition was not significantly altered under any of the conditions tested. In addition, the resistance to antibiotics and the biochemical characteristics which were shown to conform to the original properties of the product were evaluated under the same conditions.
  • the composition according to the invention is useful in the pharmaceutical, veterinary and / or nutrition fields.
  • Enterogermina ® commercially, in particular the composition of the invention has a beneficial action on the intestine and on the immune system and is particularly suitable for treatment and prevention intestinal dysmicrobism and endogenous dysvitaminosis as well as coadjuvant treatment in the recovery of altered intestinal microbial flora following antibiotic therapy and chemotherapy, as it is suitable for example for its use in combination with antibiotics to combat YHélicobacter pylori.
  • the invention also relates to a medicament containing the composition of spores of bacteria as defined above.
  • a 600 ml pre-fermentation of a suspension of four strains of Bacillus clausii 1-273, 1-274, 1-275 and 1-276 (in equal proportions) is carried out at a concentration of 500 million spores / ml in 30 liters of fermentation medium for 7 hours.
  • the pre-fermented suspension is inoculated into 1000 liters of fermentation medium based on peptones and mineral salts; incubated at 37 ° C for 48-72 hours under aerobic conditions, the cells are separated by centrifugation from the culture medium with distilled water until a final volume of 100 liters is obtained and then the suspension is pasteurized at 70 ° C for 30 minutes.
  • a concentrated suspension is thus obtained containing a concentration of Bacillus clausii spores of about 20 billion per gram.
  • aqueous mixture of spores of Bacillus clausii 1-273, 1-274, 1-275 and 1-276 (concentrated suspension prepared as above) at the concentration of 20 billion spores per gram.
  • the concentrated suspension of spores is therefore sprayed onto the heated mixture with a pressure of 2 bar and a flow rate of 135 ml / minute while keeping the mixture in suspension with air at 60 ° C. About 110 minutes after the system stops, it is allowed to cool and the composition is recovered.
  • a composition is thus obtained having the following characteristics:
  • Example 2 To 240 g of a composition of Example 1, 57 g of microcrystalline cellulose and 3 g of magnesium stearate are added. After having been mixed, the composition thus obtained is distributed in sealed gelatin capsules of format 1, each containing 300 mg of the following composition: Matrix (Kaolin + microcrystalline cellulose) containing approximately 2 billion Bacillus Clausii spores (1-273, 1-274, 1-275, 1-276) mg 240.00
  • Matrix (Kaolin 4- Microcrystalline cellulose) containing approximately 2 billion Bacillus Clausii spores (1-273, 1-274, 1-275, 1-276) mg 200.00 *
  • Matrix (Calcium carbonate + Microcrystalline cellulose) containing approximately 2 billion Bacillus Clausii spores (1-273, 1-274, 1-275, I- mg 200.00 *

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

The invention relates to a composition of Bacillus-type non-pathogenic bacterial spores which are adsorbed on a matrix formed by at least one water-insoluble adsorbent compound and a cellulose derivative. The inventive composition can be obtained using the air-fluidised bed technique and said composition is suitable for use in the pharmaceutical, veterinary and nutritional fields.

Description

"Composition solide contenant des spores de bactéries non pathogènes du genre Bacillus" "Solid composition containing spores of non-pathogenic bacteria of the genus Bacillus"
La présente invention concerne une composition solide contenant des spores de bactéries non pathogènes, pour utilisation dans les domaines pharmaceutique, vétérinaire ou de la nutrition, notamment une composition contenant des spores de bactéries du genre Bacillus.The present invention relates to a solid composition containing spores of non-pathogenic bacteria, for use in the pharmaceutical, veterinary or nutritional fields, in particular a composition containing spores of bacteria of the genus Bacillus.
La formulation des spores aux fins de leur administration chez l'homme ou chez l'animal est difficile et problématique dès lors que, s 'agissant de matériel vivant, si l'on adopte des procédés non appropriés on court le risque de perdre une partie considérable de principe actif.The formulation of spores for the purpose of their administration in humans or animals is difficult and problematic since, in the case of living material, if we adopt unsuitable methods there is the risk of losing part considerable active ingredient.
Différentes compositions solides à base de spores sont connues. Elles sont réalisées selon différentes techniques, telles que, par exemple, la lyophilisation ou le "spray drying", de même que sont connues des suspensions liquides de spores ou de simples mélanges de spores avec des excipients conventionnels de la technique pharmaceutique, telles que par exemple WO 99/49877, où sont décrites des compositions de spores de bacilles produisant de l'acide lactique pour la réduction du cholestérol.Various solid compositions based on spores are known. They are produced according to different techniques, such as, for example, lyophilization or "spray drying", as are known liquid suspensions of spores or simple mixtures of spores with conventional excipients in the pharmaceutical technique, such as by example WO 99/49877, which describes compositions of bacillus spores producing lactic acid for the reduction of cholesterol.
L'un des inconvénients principaux de l'art antérieur est la faible stabilité des compositions qui en résultent, facteur critique qui ne permet pas le stockage pendant des périodes raisonnablement longues et qui rend nécessaire le stockage et la conservation de la composition avant son emploi à de basses températures afin de garder autant que possible inaltérées les propriétés des spores.One of the main drawbacks of the prior art is the low stability of the resulting compositions, a critical factor which does not allow storage for reasonably long periods and which makes it necessary to store and preserve the composition before its use. low temperatures to keep the spore properties as unaltered as possible.
Un autre inconvénient des compositions de l'art antérieur est la faible concentration de principe actif, de spores donc, qui peut être introduite dans la composition même.Another drawback of the compositions of the prior art is the low concentration of active principle, therefore of spores, which can be introduced into the composition itself.
Des compositions liquides à base de spores sont disponibles sur le marché, par exemple la suspension de spores de bacilles commercialisée en Italie depuis longtemps sous la marque « EnterogerrninaV Pour autant qu'elle soit efficace et appréciée depuis longtemps par les consommateurs, cette suspension ne peut pas contenir une concentration très élevée de principe actif (supérieure à 2 milliards de spores/5 ml).Liquid compositions based on spores are available on the market, for example the suspension of bacillus spores marketed in Italy for a long time under the brand name “EnterogerrninaV. As long as it is effective and appreciated for a long time by consumers, this suspension cannot not contain a very high concentration of active ingredient (greater than 2 billion spores / 5 ml).
Le but de la présente invention est de fournir une composition solide à base de spores de bactéries non pathogènes, ci-après dénommées simplement « spores », qui soit simple à produire, facile à stocker et qui contienne une quantité élevée de principe actif et stable dans le temps.The object of the present invention is to provide a solid composition based on spores of non-pathogenic bacteria, hereinafter simply called "Spores", which is simple to produce, easy to store and which contains a high quantity of active principle and stable over time.
Il a été maintenant trouvé qu'en faisant adsorber les spores sur une matrice appropriée, en utilisant la technique du lit d'air fluidisé, on obtient une forme solide de spores à très haute concentration, facile à traiter au plan industriel et très stable.It has now been found that by adsorbing the spores on an appropriate matrix, using the fluidized air bed technique, a solid form of spores is obtained at very high concentration, easy to process industrially and very stable.
Notamment, il a été trouvé que cette forme solide, ci-après dénomméeIn particular, it has been found that this solid form, hereinafter referred to as
« composition », permet de fixer à la matrice une quantité importante de spores en créant une composition solide à haute concentration dont la granulométrie et la surface spécifique la rendent particulièrement adaptée à la libération in vivo des spores tout au long du tractus gastro-intestinal."Composition" makes it possible to fix a large quantity of spores to the matrix by creating a solid composition at high concentration whose particle size and specific surface make it particularly suitable for the in vivo release of spores throughout the gastrointestinal tract.
De plus, il a été trouvé que la composition est dotée d'excellentes propriétés de fluidité et, de ce fait, elle est susceptible d'être traitée industriellement aux fins de sa formulation en compositions monodose ou multidose, sans qu'il soit nécessaire de la soumettre à des traitements ultérieurs.In addition, it has been found that the composition has excellent fluidity properties and, therefore, it is capable of being industrially treated for the purposes of its formulation in single-dose or multi-dose compositions, without it being necessary to subject it to further processing.
Ainsi, selon un de ses aspects l'invention concerne une composition de spores de bactéries non pathogènes du genre Bacillus adsorbées sur une matrice formée d'au moins un composé insoluble dans l'eau et d'un dérivé de la cellulose, susceptible être obtenue par la technique du lit d'air fluidisé.Thus, according to one of its aspects, the invention relates to a composition of spores of non-pathogenic bacteria of the genus Bacillus adsorbed on a matrix formed of at least one compound insoluble in water and of a cellulose derivative, capable of being obtained. by the fluidized air bed technique.
Plus particulièrement, l'invention concerne une composition de spores de bactéries non pathogènes du genre Bacillus susceptible d'être obtenue par un procédé qui comprend traiter selon la technique du lit d'air fluidisé une suspension liquide desdites spores avec une matrice formée d'un composé adsorbant insoluble dans l'eau et d'un dérivé de la cellulose.More particularly, the invention relates to a composition of spores of non-pathogenic bacteria of the genus Bacillus capable of being obtained by a process which comprises treating according to the technique of the fluidized air bed a liquid suspension of said spores with a matrix formed of a water-insoluble adsorbent compound and a cellulose derivative.
Selon la présente invention, l'expression « composé adsorbant » désigne un composé chimique quelconque, ou un mélange de composés chimiques, qui puisse être ingéré et soit doté de propriétés adsorbantes ; de préférence, le composé adsorbant insoluble dans l'eau est choisi dans le groupe formé d'argiles, kaolin, carbonate de calcium, silices colloïdaux, silicate de magnésium et aluminium et dérivés de la cellulose et de la bentonite, le kaolin étant particulièrement avantageux.According to the present invention, the expression “adsorbent compound” designates any chemical compound, or a mixture of chemical compounds, which can be ingested and which has adsorbent properties; preferably, the water-insoluble adsorbent compound is chosen from the group formed of clays, kaolin, calcium carbonate, colloidal silicas, magnesium silicate and aluminum and derivatives of cellulose and bentonite, kaolin being particularly advantageous .
D'après l'invention, l'expression « dérivé de la cellulose » désigne un dérivé quelconque de la cellulose qui puisse être ingéré, telles que, par exemple, la cellulose microcristalline, la méthylcellulose, l'hydroxypropyl- méthyl-cellulose, etc. dont de larges gammes commerciales sont disponibles, la cellulose microcristalline étant particulièrement préférée.According to the invention, the expression “cellulose derivative” designates any cellulose derivative which can be ingested, such as, for example, microcrystalline cellulose, methylcellulose, hydroxypropyl- methyl cellulose, etc. of which wide commercial ranges are available, microcrystalline cellulose being particularly preferred.
Il est entendu, même où cela n'est pas expressément indiqué, que tous les composants de la composition de l'invention sont du type pouvant être ingéré par l'homme et/ou l'animal, c'est à dire "non toxique". Notamment, la nature et la qualité des composants seront choisies en fonction de l'utilisation ultime de la composition et leurs genre et pureté seront donc appropriés et compatibles avec les emplois finals de la composition ; en conséquence, à titre illustratif, lorsque la composition est destinée au traitement pharmaceutique, le genre et la pureté des composants devront être acceptables au niveau pharmaceutique et ainsi de suite.It is understood, even where this is not expressly indicated, that all the components of the composition of the invention are of the type which can be ingested by humans and / or animals, that is to say "non-toxic ". In particular, the nature and quality of the components will be chosen according to the end use of the composition and their kind and purity will therefore be appropriate and compatible with the end uses of the composition; consequently, by way of illustration, when the composition is intended for pharmaceutical treatment, the type and purity of the components must be acceptable from a pharmaceutical level and so on.
Par les expressions "spores adsorbées", "adsorption" ou "adsorber" on entend, selon la présente invention, la rétention des spores à la surface de la matrice d'où ledites spores sont libérables dans le tractus digestif. Les spores peuvent être rétenues sur la matrice par n'importe quel rattachement, à savoir par une liason possible quelconque (chimique, biologique, physique,etc.) selon le type de matrice utilisée.By the expressions "adsorbed spores", "adsorption" or "adsorb" means, according to the present invention, the retention of spores on the surface of the matrix from which said spores are releasable in the digestive tract. The spores can be retained on the matrix by any attachment, namely by any possible link (chemical, biological, physical, etc.) depending on the type of matrix used.
Les quantités relatives des deux composants qui constituent la matrice peuvent varier dans une large fourchette. Par exemple, le rapport en poids entre le composé adsorbant et le dérivé de la cellulose peut être compris entreThe relative amounts of the two components that make up the matrix can vary within a wide range. For example, the weight ratio between the adsorbent compound and the cellulose derivative can be between
90 : 10 et 10 :90, de préférence entre 70 :30 et 30 :70, encore plus préférablement entre 60 :40 et 40 :60, les deux composants étant avantageusement présents dans la matrice dans un rapport en poids d'environ 50 :50. En tout état de cause, la matrice de l'invention est un mélange insoluble dans l'eau, inerte vers les spores, où par « inerte vers les spores » on entend qu'elle n'interfère pas négativement avec les spores.90: 10 and 10: 90, preferably between 70: 30 and 30: 70, even more preferably between 60: 40 and 40: 60, the two components being advantageously present in the matrix in a weight ratio of approximately 50: 50. In any event, the matrix of the invention is a mixture insoluble in water, inert towards the spores, where by "inert towards the spores" is meant that it does not interfere negatively with the spores.
Les spores pouvant être employées dans la présente invention sont de préférence des spores de bactéries non pathogènes particulièrement utiles dans les domaines pharmaceutique, vétérinaire et/ou de la nutrition. La composition de la présente invention peut contenir des spores d'un seul Bacillus ou des spores de différents Bacilles mélangés.The spores which can be used in the present invention are preferably spores of non-pathogenic bacteria which are particularly useful in the pharmaceutical, veterinary and / or nutritional fields. The composition of the present invention may contain spores of a single Bacillus or spores of different Bacilli mixed.
Selon un aspect préféré, la composition de l'invention comprend des spores de Bacillus subtilis ou de Bacillus clausii. De préférence, la composition de l'invention comprend les spores d'une ou plusieurs souches de Bacillus clausii (dont la dénomination taxonomique antérieure était Bacillus subtilis), déposées en conformité au Traité deAccording to a preferred aspect, the composition of the invention comprises spores of Bacillus subtilis or Bacillus clausii. Preferably, the composition of the invention comprises the spores of one or more strains of Bacillus clausii (whose previous taxonomic name was Bacillus subtilis), deposited in accordance with the Treaty of
Budapest auprès du CNCM Institut Pasteur sous les numéros d'ordre : 1-273, 1-274, 1-275, 1-276.Budapest at the CNCM Institut Pasteur under order numbers: 1-273, 1-274, 1-275, 1-276.
Pour la préparation de la composition, on utilise la technique du lit d'air fluidisé bien connue à homme du métier.For the preparation of the composition, the fluidized air bed technique well known to those skilled in the art is used.
Selon cette technique, on pulvérise une suspension aqueuse contenant les spores, ci-après dénommée « suspension concentrée », sur une matrice obtenue en mélangeant au moins un composé adsorbant et un dérivé de la cellulose, agitée constamment par un flux d'air pendant toute la durée du procédé, dans un outillage prévu à cet effet, tel que, par exemple, une machine à granuler au lit d'air fluidisé.According to this technique, an aqueous suspension containing the spores, hereinafter called "concentrated suspension", is sprayed onto a matrix obtained by mixing at least one adsorbent compound and a cellulose derivative, constantly agitated by a flow of air throughout the duration of the process, in a tool provided for this purpose, such as, for example, a machine for granulating with a fluidized air bed.
La suspension concentrée est aqueuse qui présente une très haute concentration en spores.The concentrated suspension is aqueous which has a very high concentration of spores.
Selon un aspect particulièrement avantageux de l'invention, la suspension concentrée est obtenue par inoculation d'une ou plusieurs souches de Bacilles dans un milieu de culture (par exemple à base de peptones et sels minéraux), en incubant le mélange à une température appropriée pendant 48-72 heures en conditions aérobies, en séparant par centrifugation les cellules du milieu épuisé avec de l'eau distillée et ensuite en pasteurisant la suspension obtenue. Un exemple de telle préparation est donné dans la partie expérimentale.According to a particularly advantageous aspect of the invention, the concentrated suspension is obtained by inoculation of one or more strains of Bacilli in a culture medium (for example based on peptones and mineral salts), by incubating the mixture at an appropriate temperature for 48-72 hours under aerobic conditions, by centrifuging the cells from the spent medium with distilled water and then pasteurizing the suspension obtained. An example of such a preparation is given in the experimental part.
La suspension concentrée ainsi préparée a une concentration de spores de Bacillus supérieure à 10 milliards par gramme, normalement comprise entre environ 15 et environ 25 milliards par gramme ou même plus.The concentrated suspension thus prepared has a Bacillus spore concentration greater than 10 billion per gram, normally between about 15 and about 25 billion per gram or even more.
D'une façon générale, la suspension concentrée est préférablement conservée après avoir été congelée à cause de son instabilité élevée à la température ambiante. Dans ce cas particulier, la suspension concentrée sera décongelée juste avant son emploi dans le procédé de la présente invention. Alternativement, la suspension concentrée peut être une suspension extemporanée de spores conservées sous forme lyophilisée dans l'eau.Generally, the concentrated suspension is preferably stored after being frozen because of its high instability at room temperature. In this particular case, the concentrated suspension will be thawed just before its use in the process of the present invention. Alternatively, the concentrated suspension can be an extemporaneous suspension of spores preserved in lyophilized form in water.
La température du flux d'air utilisé dans le procédé de la présente invention est comprise entre la température ambiante et la température maximale supportée par les spores ; selon un aspect préféré, le procédé a lieu de préférence à une température comprise entre 40° et 90 °C, avantageusement entre 60 et 80 °C.The temperature of the air flow used in the process of the present invention is between ambient temperature and the maximum temperature supported by the spores; according to a preferred aspect, the process takes place preferably at a temperature between 40 ° and 90 ° C, advantageously between 60 and 80 ° C.
Une fois achevée la pulvérisation et l' adsorption de la matrice concentrée, la composition obtenue est ultérieurement gardée en suspension au moyen du flux d'air chauffé jusqu'à ce qu'il ait atteint l'humidité résiduelle souhaitée, de préférence, jusqu'à ce que l'humidité soit inférieure à 3%, avantageusement inférieure ou égale à 2%.Once spraying and adsorption of the concentrated matrix have been completed, the composition obtained is subsequently kept in suspension by means of the flow of heated air until it has reached the desired residual humidity, preferably up to the humidity is less than 3%, advantageously less than or equal to 2%.
La durée du processus est définie par la quantité de suspension concentrée à pulvériser, par la vitesse de pulvérisation ainsi que par la température du flux d'air. Normalement, pour le traitement de quantités autour de 30 kg de matrice, deux heures environ sont nécessaires pour achever le procédé.The duration of the process is defined by the quantity of concentrated suspension to be sprayed, by the spraying speed as well as by the temperature of the air flow. Normally, for the treatment of quantities around 30 kg of matrix, approximately two hours are necessary to complete the process.
Le procédé de préparation de la composition constitue un objet ultérieur de la présente invention.The process for preparing the composition constitutes a further subject of the present invention.
Ainsi que cela est indiqué ci-dessus, la composition de l'invention peut être obtenue sous une forme hautement concentrée et présenter une concentration comprise, par exemple, entre 3 et 30 milliards de spores par gramme de composition finale, par exemple entre 5 et 20 milliards de spores par gramme de composition finale, avantageusement d'environ 10 milliards de spores par gramme, permettant ainsi l'administration de quantités consistantes de spores dans des petits volumes. Cette importante propriété fait que la composition est particulièrement facile à utiliser et, par exemple, peut être introduite dans de petites gélules ou sachets ou incorporée dans des aliments ou dans d'autres compositions. Au besoin, la composition peut également être administrée après avoir été mise à nouveau en suspension dans de l'eau ou dans d'autres liquides appropriés.As indicated above, the composition of the invention can be obtained in a highly concentrated form and have a concentration of, for example, between 3 and 30 billion spores per gram of final composition, for example between 5 and 20 billion spores per gram of final composition, advantageously about 10 billion spores per gram, thus allowing the administration of consistent amounts of spores in small volumes. This important property makes the composition particularly easy to use and, for example, can be introduced into small capsules or sachets or incorporated into food or other compositions. If necessary, the composition can also be administered after being resuspended in water or other suitable liquids.
La composition de l'invention s'est avérée stable tout en gardant inaltéré son titre pendant longtemps, même à des températures supérieures à la température ambiante (environ 40 °C).The composition of the invention has been found to be stable while retaining its title unchanged for a long time, even at temperatures above ambient temperature (approximately 40 ° C.).
La détermination du titre en spores de la composition de l'invention peut être menée selon un mode opératoire quelconque, par exemple par comptage sur des plaques de culture conventionnelles.The determination of the spore titer of the composition of the invention can be carried out according to any procedure, for example by counting on conventional culture plates.
Comme indiqué ci-dessus, la composition de l'invention présente une large surface spécifique, grâce à une taille des particules très fine, jusqu'à atteindreAs indicated above, the composition of the invention has a large specific surface, thanks to a very fine particle size, until it reaches
90% des particules de la composition dont la taille des particules est inférieure ou égale à 130 micromètres, de préférence avec 60% des particules de la composition dont la taille des particules est inférieure ou égale à 60 micromètres.90% of the particles of the composition whose particle size is less than or equal to 130 micrometers, preferably with 60% of the particles of the composition whose particle size is less than or equal to 60 micrometers.
De plus, la composition n'a pas d'odeur ni de saveur et peut être ainsi ajoutée à des aliments, boissons, ou à d'autres compositions sans altérer leur saveur originale.In addition, the composition has no odor or flavor and can thus be added to foods, drinks, or other compositions without altering their original flavor.
Si l'on veut, la composition peut contenir des additifs opportunément choisis en fonction du consommateur final, de la modalité d'absorption ou du genre de traitement ultérieur auquel on souhaite la soumettre, à l'unique condition que les additifs soient inertes versus les spores. Par exemple, il sera possible d'ajouter des agents lubrifiants, diluants, etc. ou tout autre agent susceptible d'en accroître l'écoulement ou d'exalter d'autres propriétés physiques particulières dans le but de faciliter les traitements ultérieurs de la composition.If desired, the composition may contain additives suitably chosen according to the final consumer, the method of absorption or the type of subsequent treatment to which it is desired to subject it, on the sole condition that the additives are inert versus the spores. For example, it will be possible to add lubricants, thinners, etc. or any other agent capable of increasing the flow thereof or of enhancing other particular physical properties in order to facilitate the subsequent treatments of the composition.
Par exemple, au cas où on souhaiterait introduire la composition dans des capsules de gélatine dure, il pourrait être utile d'ajouter du stéarate de magnésium et/ou de la cellulose microcristalline.For example, if it is desired to introduce the composition into hard gelatin capsules, it could be useful to add magnesium stearate and / or microcrystalline cellulose.
Alternativement ou conjointement aux additifs précités, pourront être ajoutés des aromatisants susceptibles de conférer à la composition des parfums ou des saveurs particuliers. Les composants ultérieurs éventuels peuvent être ajoutés à la matrice avant l' adsorption des spores ou tout simplement à la composition finale obtenue par le procédé de l'invention.Alternatively or in conjunction with the aforementioned additives, flavoring agents may be added which may impart particular fragrances or flavors to the composition. Any subsequent components can be added to the matrix before adsorption of the spores or simply to the final composition obtained by the process of the invention.
Au besoin, la composition peut faire l'objet de modifications ultérieures ; la composition peut donc être granulée selon les techniques bien connues au cas où on souhaiterait procéder à sa compression, ou être traitée de façon à obtenir des compositions à libération contrôlée selon les techniques bien connues, afin de modifier le temps de son relargage dans l'intestin.If necessary, the composition can be subject to subsequent modifications; the composition can therefore be granulated according to well known techniques if it is desired to compress it, or be treated so as to obtain controlled-release compositions according to well known techniques, in order to modify the time of its release in the intestine.
La composition peut être administrée en quantités variables selon les besoins pour lesquels elle est administrée. D'une façon générale, en cas d'administration chez l'homme, on peut prévoir 10 milliards de spores/jour et même davantage, avantageusement de 1 à 8 milliards par jour, par exemple 2, 4 ou 6 milliards de spores par jour, l'administration étant possible en une seule prise ou de façon réitérée.The composition can be administered in varying amounts depending on the needs for which it is administered. Generally, when administered to humans, one can provide 10 billion spores / day and even more, advantageously from 1 to 8 billion per day, for example 2, 4 or 6 billion spores per day , administration being possible in a single take or repeatedly.
Aux fins de son administration, la composition de l'invention peut, le cas échéant, être formulée en unités de dosage ; par exemple, grâce à ses qualités, elle peut être facilement formulée en capsules de gélatine, telles que les gélules dans le format 0, 1 ou 2, choisies par l'expert de la branche selon le dosage.For the purposes of its administration, the composition of the invention can, where appropriate, be formulated in dosage units; for example, thanks to its qualities, it can be easily formulated in gelatin capsules, such as capsules in the 0, 1 or 2 format, chosen by the sector expert according to the dosage.
Les unités de dosage, sous forme de gélules ou de sachets, contenant la composition de l'invention représentent un objet ultérieur de la présente invention.The dosage units, in the form of capsules or sachets, containing the composition of the invention represent a further object of the present invention.
Par exemple, ces unités de dosage peuvent contenir de 1 à 10 milliards desdites spores, avantageusement de 2 à 5 milliards, de 50 à 500 mg, par exemple de 50 à 250 mg de kaolin et par exemple de 50 à 600 mg, par exemple de 50 à 300 mg de cellulose microcristalline.For example, these dosage units can contain from 1 to 10 billion of said spores, advantageously from 2 to 5 billion, from 50 to 500 mg, for example from 50 to 250 mg of kaolin and for example from 50 to 600 mg, for example from 50 to 300 mg of microcrystalline cellulose.
Ces unités de dosage peuvent être administrées une ou plusieurs fois par jour, selon la nécessité et la concentration de l'unité de dosage.These dosage units can be administered one or more times per day, depending on the need and concentration of the dosage unit.
Par exemple, on peut préparer des unité de dosage contenant 5-7 milliards de spores, avantageusement environ 6 milliards de spores, et administrer ladite unité de dosage une seule fois par jour.For example, one can prepare dosage units containing 5-7 billion spores, preferably about 6 billion spores, and administer said dosage unit only once a day.
S 'agissant de matériel vivant, il est évident que le titre microbiologique peut subir des variations; par consequant un excès de spores de 10-20% par rapport à la dose prévue est préférablement rajouté dans la préparation de la composition. Compte-tenu de l'absence de toxicité du produit, un tel excès ne cause toutefois aucun problème.As regards living material, it is obvious that the microbiological title can undergo variations; therefore an excess of spores of 10-20% relative to the planned dose is preferably added in the preparation of the composition. Considering the absence of toxicity of the product, such an excess does not however cause any problem.
Certains lots de la compositions de l'invention conditionnés dans le verre ou dans le polyéthylène ont été soumis à des études de stabilité pour en évaluer le comportement à des températures différentes (de 5°C à 40 °C) et dégrés d'humidité (jusqu'à 75% d'humidité relative). Les résultats après 24 mois ont montré que le titre de la composition n'a pas été altérée d'une façon significative dans aucune des conditions expérimentées. En outre on a évalué aux mêmes conditions la résistance aux antibiotiques et les caractéristiques biochimiques qui se sont montrées conformes aux propriétés originales du produit. La composition selon l'invention est utile dans les domaines pharmaceutique, vétérinaire et/ou de la nutrition.Certain batches of the composition of the invention packaged in glass or in polyethylene were subjected to stability studies to evaluate their behavior at different temperatures (from 5 ° C. to 40 ° C.) and degrees of humidity ( up to 75% relative humidity). The results after 24 months showed that the titer of the composition was not significantly altered under any of the conditions tested. In addition, the resistance to antibiotics and the biochemical characteristics which were shown to conform to the original properties of the product were evaluated under the same conditions. The composition according to the invention is useful in the pharmaceutical, veterinary and / or nutrition fields.
Elle possède notamment les mêmes applications que le produitIn particular, it has the same applications as the product
Enterogermina® dans le commerce, en particulier la composition de l'invention exerce une action bénéfique sur l'intestin et sur le système immunitaire et est particulièrement adaptée pour le traitement et la prévention du dysmicrobisme intestinal et la dysvitaminose endogène ainsi qu'en traitement coadjuvant dans la récupération de la flore microbienne intestinale altérée suite à la thérapie antibiotique et à la chimiothérapie, ainsi qu'elle est adaptée par exemple pour son utilisation en association avec des antibiotiques pour combattre YHélicobacter pylori .Enterogermina ® commercially, in particular the composition of the invention has a beneficial action on the intestine and on the immune system and is particularly suitable for treatment and prevention intestinal dysmicrobism and endogenous dysvitaminosis as well as coadjuvant treatment in the recovery of altered intestinal microbial flora following antibiotic therapy and chemotherapy, as it is suitable for example for its use in combination with antibiotics to combat YHélicobacter pylori.
L'invention a également par objet un médicament contenant la composition de spores de bactéries telle que définie ci-dessus.The invention also relates to a medicament containing the composition of spores of bacteria as defined above.
Les exemples indiqués ci-après illustrent l'invention sans pour autant la limiter. PREPARATION DE LA SUSPENSION CONCENTREEThe examples indicated below illustrate the invention without however limiting it. PREPARATION OF THE CONCENTRATED SUSPENSION
On conduit une pré-fermentation de 600 ml d'une suspension de quatre souches de Bacillus clausii 1-273, 1-274, 1-275 et 1-276 (en égales proportions) à la concentration de 500 millions de spores/ml dans 30 litres de milieu de fermentation pendant 7 heures. On inocule la suspension pre- fermentée dans 1000 litres de milieu de fermentation à base de peptones et sels minéraux; on incube à 37°C pendant 48-72 heures en conditions aérobies, on sépare par centrifugation les cellules du milieu de culture avec de l'eau distillée jusqu'à obtenir un volume final de 100 litres et ensuite on pasteurise la suspension à 70 °C pendant 30 minutes. On obtient ainsi une suspension concentrée contenant une concentration de spores de Bacillus clausii d'environ 20 milliards par gramme. EXEMPLE 1A 600 ml pre-fermentation of a suspension of four strains of Bacillus clausii 1-273, 1-274, 1-275 and 1-276 (in equal proportions) is carried out at a concentration of 500 million spores / ml in 30 liters of fermentation medium for 7 hours. The pre-fermented suspension is inoculated into 1000 liters of fermentation medium based on peptones and mineral salts; incubated at 37 ° C for 48-72 hours under aerobic conditions, the cells are separated by centrifugation from the culture medium with distilled water until a final volume of 100 liters is obtained and then the suspension is pasteurized at 70 ° C for 30 minutes. A concentrated suspension is thus obtained containing a concentration of Bacillus clausii spores of about 20 billion per gram. EXAMPLE 1
Dans un système pour le procédé à lit d'air fluidisé, on charge 15 kg de kaolin de qualité pharmaceutique et 15 kg de cellulose microcristalline de qualité pharmaceutique et on chauffe le mélange pendant quelques minutes au moyen d'un flux d'air à 60°C. Sous une hotte au flux laminaire, on charge dans un contenant doté d'une pompe péristaltique reliée à des buses de nébulisation de 1,2 mm de diamètre et reliées à l'installation à lit d'air fluidisé, 15 kg d'une suspension aqueuse d'un mélange de spores de Bacillus clausii 1-273, 1-274, 1-275 et 1-276 (suspension concentrée préparée comme ci-dessus) à la concentration de 20 milliards de spores par gramme. On pulvérise donc la suspension concentrée de spores sur le mélange chauffé avec une pression de 2 bar et un débit de 135 ml/minute en gardant en suspension le mélange avec de l'air à 60 °C. Environ 110 minutes après on arrête le système, on laisse refroidir et on récupère la composition. On obtient ainsi une composition présentant les caractéristiques suivantes :In a system for the fluidized air bed process, 15 kg of pharmaceutical grade kaolin and 15 kg of microcrystalline cellulose of pharmaceutical quality are loaded and the mixture is heated for a few minutes by means of an air flow at 60 ° C. Under a laminar flow hood, 15 kg of a suspension is loaded into a container equipped with a peristaltic pump connected to nebulization nozzles of 1.2 mm in diameter and connected to the installation with a fluidized air bed. aqueous mixture of spores of Bacillus clausii 1-273, 1-274, 1-275 and 1-276 (concentrated suspension prepared as above) at the concentration of 20 billion spores per gram. The concentrated suspension of spores is therefore sprayed onto the heated mixture with a pressure of 2 bar and a flow rate of 135 ml / minute while keeping the mixture in suspension with air at 60 ° C. About 110 minutes after the system stops, it is allowed to cool and the composition is recovered. A composition is thus obtained having the following characteristics:
- titre - déterminé par comptage sur plaque: 10 milliards de spores/g ; - humidité résiduelle < 2% ;- title - determined by counting on a plate: 10 billion spores / g; - residual humidity <2%;
- granulométrie - déterminée par un granulomètre laser Malvern9 dans de l'eau déminéralisée: 90% des particules < 100 μm- granulometry - determined by a Malvern 9 laser granulometer in demineralized water: 90% of the particles <100 μm
60% des particules < 50 μm60% of particles <50 μm
- surface spécifique: 3-5 m2/g. EXEMPLE 2- specific surface: 3-5 m 2 / g. EXAMPLE 2
En opérant comme dcrit dans l'Exemple 1, mais en utilisant seulement la souche 1-274 de Bacillus clausii, on obtient un produit ayant les caractéristiques suivantes:By operating as described in Example 1, but using only the Bacillus clausii strain 1-274, a product is obtained having the following characteristics:
- titre - déterminé par comptage sur plaque : 10 milliards de spores/g ; - humidité résiduelle <_ 2% ;- title - determined by counting on a plate: 10 billion spores / g; - residual humidity <_ 2%;
- granulométrie - déterminée par un granulomètre laser Malvern* dans de l'eau déminéralisée: 90% des particules < 130 μm- granulometry - determined by a Malvern * laser granulometer in demineralized water: 90% of particles <130 μm
60% des particules < 60 μm60% of particles <60 μm
- surface spécifique: 3,5-5 m2/g. EXEMPLE 3- specific surface: 3.5-5 m 2 / g. EXAMPLE 3
En opérant comme décrit dans l'Exemple 1, mais en utilisant seulement la souche 1-276 de Bacillus clausii , on obtient un produit ayant les caractéristiques suivantes:By operating as described in Example 1, but using only strain 1-276 of Bacillus clausii, a product is obtained having the following characteristics:
- titre de 12 milliards de spores/g ; - humidité résiduelle <_ 3 % ;- titer of 12 billion spores / g; - residual humidity <_ 3%;
- granulométrie - déterminée par un granulomètre laser Malvern8 dans de l'eau déminéralisée: 90% des particules < 130 μm- granulometry - determined by a Malvern 8 laser granulometer in demineralized water: 90% of particles <130 μm
60% des particules < 60 μm60% of particles <60 μm
- surface spécifique: 3-5 m2/g. EXEMPLE 4- specific surface: 3-5 m 2 / g. EXAMPLE 4
A 240 g d'une composition de l'Exemple 1 on ajoute 57 g de cellulose microcristalline et 3 g de stéarate de magnésium. Après avoir été mélangée, la composition ainsi obtenue est distribuée dans des capsules operculées de gélatine dure de format 1, contenant chacune 300 mg de la composition suivante : Matrice (Kaolin + Cellulose microcristalline) contenant environ 2 milliards de spores de Bacillus Clausii (1-273, 1-274, 1-275, 1-276) mg 240,00To 240 g of a composition of Example 1, 57 g of microcrystalline cellulose and 3 g of magnesium stearate are added. After having been mixed, the composition thus obtained is distributed in sealed gelatin capsules of format 1, each containing 300 mg of the following composition: Matrix (Kaolin + microcrystalline cellulose) containing approximately 2 billion Bacillus Clausii spores (1-273, 1-274, 1-275, 1-276) mg 240.00
Cellulose microcristalline mg 57,00Microcrystalline cellulose mg 57.00
Stéarate de magnésium mg 3,00Magnesium stearate mg 3.00
EXEMPLE 5EXAMPLE 5
CAPSULES DE GELATINE DUREHARD GELATIN CAPSULES
FORMAT 1 CONTENANT 275 mg DE COMPOSITIONFORMAT 1 CONTAINING 275 mg OF COMPOSITION
Matrice (Kaolin 4- Cellulose microcristalline) contenant environ 2 milliards de spores de Bacillus Clausii (1-273, 1-274, 1-275, 1-276) mg 200,00*Matrix (Kaolin 4- Microcrystalline cellulose) containing approximately 2 billion Bacillus Clausii spores (1-273, 1-274, 1-275, 1-276) mg 200.00 *
Cellulose microcristalline mg 72,25Microcrystalline cellulose mg 72.25
Stéarate de magnésium d'origine végétale mg 2,75Plant-based magnesium stearate mg 2.75
(* dans la fomule de fabrication 220,00 mg correspondant à un surdosage de 10%)(* in the manufacturing formula 220.00 mg corresponding to an overdose of 10%)
EXEMPLE 6EXAMPLE 6
CAPSULES DE GELATINE DUREHARD GELATIN CAPSULES
FORMAT 1 CONTENANT 255 mg DE COMPOSITIONFORMAT 1 CONTAINING 255 mg OF COMPOSITION
Matrice (Carbonate de calcium + Cellulose microcristalline) contenant environ 2 milliards de spores de Bacillus Clausii (1-273, 1-274, 1-275, I- mg 200,00*Matrix (Calcium carbonate + Microcrystalline cellulose) containing approximately 2 billion Bacillus Clausii spores (1-273, 1-274, 1-275, I- mg 200.00 *
276)276)
Cellulose microcristalline mg 52,25Microcrystalline cellulose mg 52.25
Stéarate de magnésium d'origine végétale mg 2,75Plant-based magnesium stearate mg 2.75
(* dans la fomule de fabrication 220,00 mg correspondant à un surdosage de (* in the manufacturing formula 220.00 mg corresponding to an overdose of

Claims

REVENDICATIONS
1. Composition de spores de bactéries non pathogènes du genre Bacillus adsorbées sur une matrice formée d'au moins un composé adsorbant insoluble dans l'eau et d'un dérivé de la cellulose, susceptible d'être obtenue par la technique du lit d'air fluidisé.1. Composition of spores of non-pathogenic bacteria of the genus Bacillus adsorbed on a matrix formed of at least one adsorbent compound insoluble in water and of a cellulose derivative, capable of being obtained by the bed technique. fluidized air.
2. Composition selon la revendication 1, caractérisée en ce qu'elle est susceptible d'être obtenue par un procédé qui comprend traiter, selon la technique du lit d'air fluidisé, une suspension liquide desdites spores avec une matrice formée d'au moins un composé adsorbant insoluble dans l'eau et d'un dérivé de la cellulose.2. Composition according to claim 1, characterized in that it is capable of being obtained by a process which comprises treating, according to the technique of the fluidized air bed, a liquid suspension of said spores with a matrix formed of at least a water-insoluble adsorbent compound and a cellulose derivative.
3. Composition selon les revendications 1 ou 2, caractérisée en ce qu'elle contient de 3 à 30 milliards desdites spores par gramme de composition.3. Composition according to claims 1 or 2, characterized in that it contains from 3 to 30 billion of said spores per gram of composition.
4. Composition selon la revendication 3, caractérisée en ce qu'elle contient de 5 à 20 milliards desdites spores par gramme de composition. 4. Composition according to claim 3, characterized in that it contains from 5 to 20 billion of said spores per gram of composition.
5. Composition selon la revendication 4, caractérisée en ce qu'elle contient environ 10 milliards desdites spores par gramme de composition.5. Composition according to claim 4, characterized in that it contains approximately 10 billion of said spores per gram of composition.
6. Composition selon l'une des revendications de 1 à 5, caractérisée en ce que ledit composé adsorbant est le kaolin.6. Composition according to one of claims from 1 to 5, characterized in that said adsorbent compound is kaolin.
7. Composition selon l'une des revendications de 1 à 5, caractérisée en ce que ledit composé adsorbant est le carbonate de calcium.7. Composition according to one of claims from 1 to 5, characterized in that said adsorbent compound is calcium carbonate.
8. Composition selon l'une des revendications de 1 à 7, caractérisée en ce que ledit dérivé de la cellulose est la cellulose microctristalline.8. Composition according to one of claims from 1 to 7, characterized in that said cellulose derivative is microctristalline cellulose.
9. Composition selon l'une des revendications de 1 à 8, caractérisée en ce que le rapport en poids entre ledit composé adsorbant et ledit dérivé de la cellulose est compris entre 90 : 10 et 10 :90.9. Composition according to one of claims from 1 to 8, characterized in that the weight ratio between said adsorbent compound and said cellulose derivative is between 90: 10 and 10: 90.
10. Composition selon la revendication 9 caractérisée en ce que ledit rapport est compris entre 70 :30 et 30 :70.10. Composition according to claim 9 characterized in that said ratio is between 70:30 and 30:70.
11. Composition selon la revendication 11 caractérisée en ce que ledit rapport est environ 50 :50. 11. Composition according to claim 11 characterized in that said ratio is approximately 50: 50.
12. Composition selon l'une des revendications de 1 à 11 sous forme d'unité de dosage en gélules ou sachets. 12. Composition according to one of claims from 1 to 11 in the form of dosage unit in capsules or sachets.
13. Composition selon la revendication 12, contenant pour chaque unité de dosage de 1 à 10 milliards desdites spores, de 50 à 500 mg de kaolin et de 50 à 600 mg de cellulose microcristalline. 13. Composition according to claim 12, containing for each dosage unit from 1 to 10 billion of said spores, from 50 to 500 mg of kaolin and from 50 to 600 mg of microcrystalline cellulose.
14. Composition selon la revendication 13, contenant pour chaque unité de dosage de 2 à 5 milliards desdites spores, de 50 à 250 mg de kaolin et de 50 à 300 mg de cellulose microcristalline.14. Composition according to claim 13, containing for each dosage unit from 2 to 5 billion of said spores, from 50 to 250 mg of kaolin and from 50 to 300 mg of microcrystalline cellulose.
15. Composition selon la revendication 12, contenant pour chaque unité de dosage environ 2 milliards desdites spores en gélules.15. The composition of claim 12, containing for each dosage unit about 2 billion of said spores in capsules.
16. Composition selon la revendication 12, contenant pour chaque unité de dosage environ 6 milliards desdites spores en gélules.16. Composition according to claim 12, containing for each dosage unit approximately 6 billion of said spores in capsules.
17. Composition selon l'une des revendications 1 à 16, caractérisée en ce que lesdites spores sont des spores de Bacillus subtilis ou de Bacillus clausii. 17. Composition according to one of claims 1 to 16, characterized in that said spores are spores of Bacillus subtilis or Bacillus clausii.
18. Composition selon la revendication 17, caractérisée en ce que lesdites spores proviennent d'un ou plusieurs souches choisies parmi Bacillus clausii 1-273, I- 274, 1-275 et 1-276. 18. Composition according to claim 17, characterized in that said spores come from one or more strains chosen from Bacillus clausii 1-273, I-274, 1-275 and 1-276.
19. Composition selon l'une des revendications 1 à 16, caractérisée en ce que lesdites spores sont un mélange de spores de Bacilles différents. 19. Composition according to one of claims 1 to 16, characterized in that said spores are a mixture of spores of different Bacilli.
20. Médicament comprenant la composition selon l'une des revendications de 1 à 19. 20. Medicament comprising the composition according to one of claims from 1 to 19.
EP02791476A 2001-07-27 2002-07-26 Solid composition containing bacillus-type non-pathogenic bacterial spores Expired - Lifetime EP1429808B1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
SI200230908T SI1429808T1 (en) 2001-07-27 2002-07-26 Solid composition containing bacillus-type non-pathogenic bacterial spores
CY20101100602T CY1112608T1 (en) 2001-07-27 2010-06-30 SOLID COMPOSITION THAT CONTAINS NON-PATHOGENIC BACTERIUM BACTERIUM SEEDS

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IT2001MI001632A ITMI20011632A1 (en) 2001-07-27 2001-07-27 SOLID COMPOSITION CONTAINING SPORE OF NON-PATHOGENIC BACTERIA OF THE GENERAL BACILLUS
ITMI20011632 2001-07-27
PCT/EP2002/008384 WO2003011341A1 (en) 2001-07-27 2002-07-26 Solid composition containing bacillus-type non-pathogenic bacterial spores

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EP1429808A1 true EP1429808A1 (en) 2004-06-23
EP1429808B1 EP1429808B1 (en) 2010-03-31

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JP (1) JP4913986B2 (en)
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AT (1) ATE462448T1 (en)
AU (1) AU2002333278B2 (en)
BR (1) BRPI0211453B1 (en)
CA (1) CA2454389C (en)
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CY (1) CY1112608T1 (en)
DE (1) DE60235819D1 (en)
DK (1) DK1429808T3 (en)
EA (1) EA006847B1 (en)
ES (1) ES2356325T3 (en)
GE (2) GEP20063721B (en)
HK (1) HK1061644A1 (en)
HR (1) HRP20040066B1 (en)
HU (1) HU229652B1 (en)
IL (2) IL159807A0 (en)
IT (1) ITMI20011632A1 (en)
MA (1) MA27051A1 (en)
MX (1) MXPA04000835A (en)
NO (1) NO332656B1 (en)
NZ (1) NZ530743A (en)
PL (1) PL209647B1 (en)
PT (1) PT1429808E (en)
SI (1) SI1429808T1 (en)
TN (1) TNSN04018A1 (en)
UA (1) UA81225C2 (en)
WO (1) WO2003011341A1 (en)
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US20120009167A1 (en) 2012-01-12
HK1061644A1 (en) 2004-09-30
US8039006B2 (en) 2011-10-18
EA200400072A1 (en) 2004-08-26
UA81225C2 (en) 2007-12-25
US8551498B2 (en) 2013-10-08
PL209647B1 (en) 2011-09-30
PT1429808E (en) 2010-06-23
PL366984A1 (en) 2005-02-07
WO2003011341A1 (en) 2003-02-13
HUP0402024A2 (en) 2005-01-28
GEP20063721B (en) 2006-01-10
ATE462448T1 (en) 2010-04-15
HRP20040066B1 (en) 2012-02-29
JP2004538311A (en) 2004-12-24
HRP20040066A2 (en) 2004-06-30
IL159807A (en) 2010-05-31
SI1429808T1 (en) 2010-11-30
NZ530743A (en) 2005-08-26
GEP20053721B (en) 2006-01-10
KR20040032868A (en) 2004-04-17
MA27051A1 (en) 2004-12-20
CN1558776A (en) 2004-12-29
CY1112608T1 (en) 2016-02-10
TNSN04018A1 (en) 2006-06-01
US20040241772A1 (en) 2004-12-02
DE60235819D1 (en) 2010-05-12
KR20090099022A (en) 2009-09-18
CN1323720C (en) 2007-07-04
MXPA04000835A (en) 2004-05-14
HUP0402024A3 (en) 2005-06-28
CA2454389C (en) 2011-07-12
ES2356325T3 (en) 2011-04-07
CA2454389A1 (en) 2003-02-13
EA006847B1 (en) 2006-04-28
IL159807A0 (en) 2004-06-20
JP4913986B2 (en) 2012-04-11
ITMI20011632A1 (en) 2003-01-27
DK1429808T3 (en) 2010-07-19
AU2002333278B2 (en) 2007-05-31
CO5560587A2 (en) 2005-09-30
BRPI0211453B1 (en) 2018-04-03
NO332656B1 (en) 2012-11-26
NO20040347L (en) 2004-03-26
ZA200400440B (en) 2005-03-30
EP1429808B1 (en) 2010-03-31
KR100970787B1 (en) 2010-07-16
ITMI20011632A0 (en) 2001-07-27
HU229652B1 (en) 2014-03-28
BR0211453A (en) 2004-08-17

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